Project 2. Preclinical Trials Abstract Non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM) are highly lethal cancers with suboptimal therapies. The goal of this Program Project and of Project 2 is to successfully implement the use of T cells expressing chimeric antigen receptors (CARs), an approach that has been highly successful in leukemias. Our group was among the first to inititate CAR T cell trials focused on solid tumors by targeting the tumor-associated antigen mesothelin. However, despite showing safety and feasibility, therapeutic efficacy was limited. We hypothesize that one of the important issues limiting success in solid tumors is tumor antigen heterogeneity. Even at baseline, it is likely that not all tumor cells will express the targeted antigen. The primary focus of this Project will be to explore ways to overcome heterogeneity in solid tumor CAR T cell therapy. Our first approach will be to attack the tumor stroma. We previously showed that CAR T cells targeted to mouse fibroblast activation protein (FAP) on tumor fibroblasts can decrease the tumor matrix, augment endogenous intratumoral T cells, and slow tumor growth with minimal toxicity. In Aim 1, we propose to study a CAR targeted to human FAP that can be used in our clinical trial. In Aim 1A, we will assess the efficacy of the anti-human-CAR T cells using a new mouse model which combines human CAFs and human tumors in immunodeficient mice. In Aim 1B, we will evaluate the safety of anti-human FAP CAR T cells on primary human cell lines. In Aim 1C, we will evaluate combination therapies using the stromal-targeted CAR (FAP-CAR) with a tumor-cell targeted CAR (mesothelin-CAR). In Aim 2, we will explore the key issue of whether mesothelin and FAP CARs are able to induce bystander effects. In Aim 2A, we will study CAR- induced bystander effects by injecting mice with varying ratio's of tumor cells expressing or not expressing mesothelin and defining bystander effect as the minimal percentage of non-targeted tumor cells that still allow tumor elimination. Additional studies are proposed to determine the mechanisms by which the bystander effect occurs. In Aim 2B, the process of epitope spreading will be studied by determining the ability of Meso- and FAP-CARs to induce or stimulate endogenous T cells directed against tumors expressing a xenoantigen, a viral antigen, and a neoantigen. We will define the amount of baseline epitope spreading that is present using tetramer assays and a T cell cross presentation proliferation assay. In Aim 3, we will explore ways in which CAR-induced antigen spreading can be augmented by immune activators such as antibodies to PD1 and CTLA-4 or an IDO inhibitor (Aim 3A) and by genetically altering CAR T cells to produce FMS-like tyrosine kinase 3 ligand (FLT3L), a powerful inducer of dendritic cells (Aim 3B). Successful completion of these studies will provide key information for the field of adoptive transfer of T cells for solid tumors (especially with regard to the very understudied issue of overcoming tumor antigen heterogeneity) and provide important data to inform the designs of the clinical trials in Project 1. 1